The broad objective of this grant application is to investigate how damaged DNA is copied by the cell's replication and repair enzymes, focusing on proteins that are induced in response to DNA damage. Damage-induced DNA repair occurs in both procaryotic and eucaryotic organisms. In Escherichia coli, response to DNA damage is orchestrated by an operon, the "SOS regulon", containing at least 25 different proteins under negative control of a repressor protein, LexA, and a multifunctional protein, RecA. In E. coli, and seemingly in animal cells as well, damage-induced DNA repair is aberrant. There is a reduction in fidelity which enables replication to continue past blocking DNA damage sites. The primary goal of this proposal is to elucidate the biochemical basis for SOS-induced error- prone repair in E. coli. Such repair depends on an activated form of RecA (RecA*) interacting with a mutagenic UmuD'2C protein complex. There are numerous types of damage occurring in DNA when cells are exposed to chemicals, drugs or radiation. To study error-prone repair in vitro and in vivo, Dr. Goodman has chosen to focus primarily on copying a site- directed abasic (apurinic/apyrimidinic) DNA lesion, a biologically relevant noncoding lesion that can occur by spontaneous and induced mechanisms. The absence of a coding base in DNA presents a strong block to replication. When replication past an abasic lesion does occur, it often results in a mutation. In this proposal, he will quantify the effects of damage-induced repair proteins in E. coli, specifically UmuD'2C, RecA*, DNA polymerase III holoenzyme, and SSB, on nucleotide insertion and excision (proofreading) at the site of the lesion, and bypass of the lesion. During the previous grant period, he succeeded in purifying UmuD'2C in soluble, active form, and showed that the complex, in the presence of RecA*, efficiently catalyzed lesion bypass. An unexpected result was that UmuD'2C catalyzed lesion bypass in the apparent absence of the polymerizing subunit of pol III holoenzyme. In other words, it is possible that UmuD'2C complex has its own low fidelity polymerase activity. If this surprising observation can be sustained, it would then have major impact in explaining how damaged DNA is copied in procaryotic and eucaryotic organisms. E. coli DNA polymerase II, is also induced in response to DNA damage, as part of the SOS regulon, but does not appear to take part in the UmuD2C - RecA* error-prone repair pathway. Another important goal is to examine the role of pol III in a UmuD'2C-independent pathway for repair of DNA damaged by exposure to ultraviolet radiation.